A specific SNP-based multiplex PCR assay for the simultaneous identification of two biological ingredients for the Chinese patent medicine, Danggui Buxue pill.

Background: An increasing number of Chinese patent medicines (CPM) have been widely used in East Asian and North American countries, and the safety and efficacy of CPM have highly attracted public attention. However, it is difficult to supervise the authenticity of multiple biological ingredients within CPM based on microscopic inspection and physical and chemical detection. The raw materials may have similar characteristics of tissue structures and ergastic substances or similar chemical composition and contents when substitutes and/or adulterants are added. DNA molecular markers have been used to distinguish the biological ingredients within CPM based on conventional PCR assay. However, it was proved to be time- and labor-consuming and reagent-wasting, as multiple PCR amplification strategies were required for identifying the complex species composition within CPM. Here, we took the CPM (Danggui Buxue pill) as an example and aimed to establish a specific SNP-based multiplex PCR assay and simultaneously determine the authenticity of the two biological ingredients (Angelicae Sinensis Radix and Astragali Radix) within this CPM. Methods: We, respectively, designed the species-specific primers based on highly variable nrITS for discriminating Angelicae Sinensis Radix and Astragali Radix from their common substitutes and adulterants. The specificity of the primers was checked through conventional PCR assay and multiplex PCR assay. Furthermore, we used a handcrafted Danggui Buxue pill sample (DGBXP) to optimize annealing temperatures for the primers with multiplex PCR, and the sensitivity was also assessed. Finally, fourteen batches of commercial Danggui Buxue pills were used to verify the stability and practicability of the established multiplex PCR assay. Results: Two pairs of highly species-specific primers for amplifying Angelicae Sinensis Radix and Astragali Radix were screened, and our established multiplex PCR assay showed high specificity and sensitivity (lowest detection concentration: 4.0 × 10-3 ng/µL) at an optimal annealing temperature of 65°C. The method could simultaneously identify both biological ingredients within the Danggui Buxue pill. Conclusion: The specific SNP-based multiplex PCR provided a simple, time-, and labor-saving method for the simultaneous identification of the two biological ingredients within Danggui Buxue pills. This study was expected to provide a novel qualitative quality control strategy for CPM.

A close look at current γδ T-cell immunotherapy.

Owing to their antitumor and major histocompatibility complex (MHC)-independent capacities, γδ T cells have gained popularity in adoptive T-cell immunotherapy in recent years. However, many unknowns still exist regarding γδ T cells, and few clinical data have been collected. Therefore, this review aims to describe all the main features of the applications of γδ T cells and provide a systematic view of current γδ T-cell immunotherapy. Specifically, this review will focus on how γδ T cells performed in treating cancers in clinics, on the γδ T-cell clinical trials that have been conducted to date, and the role of γδ T cells in the pharmaceutical industry.

Epitope identification for p53R273C mutant.

BACKGROUND: With the rise of immunotherapy based on cancer neoantigen, identification of neoepitopes has become an urgent problem to be solved. The TP53 R273C mutation is one of the hotspot mutations of TP53, however, the immunogenicity of this mutation is not yet clear. The aim of this study is to identify potential epitopes for p53R273C mutant. METHODS: In this study, bioinformatic methods, peptide exchange assay, and peptide-immunized human leukocyte antigen (HLA) transgenic mouse model were used to explore the immunogenicity of this mutation. RESULTS: Peptides with higher affinity to common HLA-A alleles (A*11:01, A*02:01) were discovered by computational prediction. All the 8-11 mer peptides contain the mutation site were synthesized and soluble peptides were used in the peptide exchange assay. However, the exchange efficiencies of these predicted peptides to HLAs were lower. Fortunately, other peptides with higher exchange efficiency were discovered. Then, the immunogenicity of these peptides was validated with the HLA-A2 transgenic mice model. CONCLUSION: We identified three potential neoepitopes of p53R273C for HLA-A*02:01, one potential neoepitope for HLA-A*11:01 and no neoepitope for HLA-A*24:02.

Safety and efficacy of mutant neoantigen-specific T-cell treatment combined anti-PD-1 therapy in stage IV solid tumors.

Aims: This trial explored the safety and efficacy of neoantigen-specific T cells (Nas-Ts) combined with anti-PD-1 (Nas-T + anti-PD-1). Patients & methods: This non-randomized trial recruited participants with solid tumors treated with at least two prior systemic treatment lines. For comparison, 1:1-matched controls who received anti-PD-1 alone were recruited. The primary end point was safety. Results: 15 participants were enrolled in the Nas-T + anti-PD-1 group, the objective response rate was 33.3%, and the disease control rate was 93.3%. The median progression-free survival was significantly different between the Nas-T + anti-PD-1 and control groups (13.8 vs 4.2 months; p = 0.024), but no difference in overall survival was found (p = 0.126). The most common adverse events were maculopapular skin reaction (53.3%), rash (53.3%), hepatotoxicity (53.3%) and fever (53.3%) in the Nas-T + anti-PD-1 group. No serious safety issues were experienced. Conclusion: Nas-Ts combined with anti-PD-1 could be more effective than anti-PD-1 alone in prolonging progression-free survival, with good safety.

Cancer immune escape is a major mechanism allowing cancer cells to avoid treatments, and PD-1 is one of those mechanisms. Nevertheless, therapies targeting PD-1 are still somewhat unsatisfactory. In this trial, we explored the safety and efficacy of mutant neoantigen-specific T cells (Nas-Ts) as adoptive cell immunotherapy individualized for each tumor, combined with an anti-PD-1 regimen (Nas-T + anti-PD-1). We recruited participants with solid tumors treated with at least two prior systemic treatment lines: 15 participants were enrolled in the Nas-T + anti-PD-1 group and 15 more in the control group. After the last follow-up, the percentage of patients on whom a therapy had some defined effect as well as the percentage of patients with advanced and metastatic cancer who achieved complete response was significantly higher for those who received Nas-T + anti-PD-1. No serious safety issues were experienced. This study confirmed that Nas-Ts combined with anti-PD-1 could be more effective than anti-PD-1 alone in delaying progression, with good safety.

Replacement of loop2 and 3 of Cry1Ai in domain II affects specificity to the economically important insect Bombyx mori.

Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity.

Construction of an anti-programmed death-ligand 1 chimeric antigen receptor and determination of its antitumor function with transduced cells.

A chimeric antigen receptor (CAR) is a type of fusion protein that comprises an antigen-recognition domain and signaling domains. In the present study, a programmed death-ligand 1 (PD-L1)-specific CAR, comprised of a single-chain variable fragment (scFv) derived from a monoclonal antibody, co-stimulatory domains of cluster of differentiation (CD) 28 and 4-1BB and a T-cell-activation domain derived from CD3ζ, was designed. The construction was cloned and packaged into the lentiviral vector pLVX. Flow cytometry confirmed that peripheral blood mononuclear cells were efficiently transduced and that the CAR was successfully expressed on T cells. The cytotoxicity of transduced T cells was detected using PD-L1-positive NCI-H358 bronchioalveolar carcinoma cells and A549 lung adenocarcinoma cells (with a low expression of PD-L1, only in the A549 cells). The results demonstrated mild cytotoxicity at an effector-to-target ratio of 10:1. An ELISA revealed a significant increase in the level of interferon-γ released from T cells transduced with scFv-28Bz when the cells were co-cultured with PD-L1-positive NCI-H358 cells, while interkeukin-2 and tumor necrosis factor-α levels remained unchanged. These data indicated a potential method for the treatment of solid tumors.

Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis).

The bacterium Bacillus thuringiensis produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two B. thuringiensis toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer (Chilo suppressalis). We found that these two proteins bind specifically to brush border membrane vesicles of C. suppressalis and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and to 428TKKMKTL434 in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the B. thuringiensis Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among B. thuringiensis toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3.

Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A548 to S798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera IMPORTANCE: Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A-C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A-C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull-down assay.

Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera.

An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

Identification of the minimal active fragment of the Cry1Ah toxin.

cry1Ah1, a novel holo-type gene cloned from Bacillus thuringiensis strain BT-8, encoded a protein exhibiting strong insecticidal activity against lepidopteran insects. To identify the minimal active fragment of the Cry1Ah toxin, 9 pairs of primers were designed to generate different PCR products. Seven PCR products were amplified by different primers using the cry1Ah1 gene as a template and cloned into a pET-21b vector. These positive clones were separately transformed into Escherichia coli. Insecticidal activity against 2nd-instar larvae of Plutella xylostella was performed using the leaf-dip bioassay: the minimal active fragment of the Cry1Ah toxin was located between amino acid residues 50I and 639E.